Pentacyclic triterpenes

ABSTRACT

The present invention relates to fungicidally effective compositions containing at least one pentacyclic triterpene compound.

[0001] This application is a divisional of copending U.S. applicationSer. No. 09/207,406 filed on Dec. 8, 1998 the disclosure of which isherein incorporated by reference.

FIELD OF THE INVENTION

[0002] The invention relates to plant protection compositions includingpentacyclic triterpenes and methods for use thereof

BACKGROUND

[0003] Outer layers of plants such as leave cuticle, fruit peels, aswell as bark protect the plant against abrasion, prevent water loss, andalso protect against pathogenic microorganisms. The breaking through theplant cuticle is a prerequisite for a pathogen to be able to enter theplants internal tissue.

[0004] The mechanism, by which plants naturally defend themselvesagainst this early stage of pathogenesis has not been fully understood.The initial process of fungal propagules attaching to a host plant isessential to the successful establishment of pathogenesis. Theestablished facts that aerial fungal pathogens bind strongly to veryhydrophobic surfaces suggests that hydrophobic forces are involved inthe attachment processes.

[0005] The attachment of aerial fungal pathogens involves an activeprocess of secretion of extracellular mucilages or adhesives, which maystart within minutes after contact with the host. Other reportedcomponents in the adhesive secretions include enzymes, among themesterases and cutinases. The erosion of cuticular waxes adjacent to andunderlying the conidium may became observable within 20 min of liquidrelease. The growth of the appressorial germ tube appears to be limitedto the zone of deposition of the liquid film.

[0006] Some studies have suggested that preparation of the infectioncourt involves active dissolution of the host cuticle by foliarpathogen. It is also believed that this dissolution is the purpose ofthese enzymes. Nicholson, R. L., and L. In: The Fungal Spore and DiseaseInitiation in Plants and Animals., Eds. Cole, G. T., and Hoch, H.C.,1991 Plenum Press, New York, 3-23. Thus, the cuticle has to bepenetrated by the attacking pathogen before the sequential steps ofdisease development occur. Some fungal spores help themselves withmechanical force exerted by the infection structure in addition to theenzymatic degradation. (Köller, W. in: The Fungal Spore and DiseaseInitiation in Plants and Animals., Eds. Cole, G. T., and Hoch, H. C.,1991 Plenum Press, New York, 219-246.

[0007] Pentacyclic triterpenes (P) are among the most common plantsecondary metabolites, but their function in plants have not beenunderstood. They are usually concentrated in the outermost layers suchas plant cuticle, fruit peel and bark. In some cases, these layerscontain very high concentration of pentacyclic triterpenes. For example,an apple peel contains about 0.1 grams of ursolic acid per fruit, andthe outer bark of white birch species contains up to 40% w/w of betulin.The amount of betulin obtainable from the birch bark waste in thewood-working industry in Finland is estimated at about 150,000 tons perannum. At present, waste bark is used as a low value fuel for energyproduction. J{umlaut over (aa)}skelin en, P. (1981) Pap. Puu 63,599-603.

[0008] Literature supplies numerous examples of enzymes that can beinhibited by PT, indicating the ability of PT to act broadly in anon-specific mode on multiple targets. See for example, (a.) Büchler etal. (1991) Biochem. Biophys. Acta 1075, 206-212, Inhibition of rat renal11 β-hydroxysteroid dehydrogenase by steroidal compounds andtriterpenoids; structure/function relationship; (b.) IKoch et al. (1994)Phytother. Res. 8, 109-111, In vitro inhibition of adenosine deaminaseby a group of steroid and triterpenoid compounds.; (c.) Najid et al.(1992) FEBS 299, 213-217, Characterization of ursolic acid as alipoxygenase and cyclooxygenase inhibitor using macrophages, plateletsand differentiated HL60 leukemic cells.; (d.) Pengsuparp et al. (1994)J. Nat. Prod. 57, 415-418, Pentacyclic triterpenes derived fromMaprounea africana are potent inhibitors of HIV-1 reversetranscriptase.; (e.) Simon et al. (1992) Biochem. Biophys. Acta 1125,68-72, Inhibition of lipoxygenase activity and HL60 leukemic cellproliferation by ursolic acid isolated from heather flowers (Callunavulgaris).; (f.) Ying et al. (1991) Biochem. J. 277, 521-526 Inhibitionof human leucocyte elastase by ursolic acid. Evidence for a binding sitefor pentacyclic triterpenes. The disclosures of each of these referencesis herein incorporated by reference.

[0009] In plant tissue cultures, stress induced by inactivated fungi orfungal enzymes has been used to enhance production of biologicallyactive secondary metabolites. In several instances it has been reportedthat this fungal elicitation led to overproduction of pentacyclictriterpenes instead of some other expected metabolites. Suitable exampleis given by Van der Heijden et al., (1988) Plant Cell Rep. 7, 51-54,where tissue cultures of Tabernaemontana spp., normally producing indolealkaloids, were subjected to stress induced by either fungi, bacteria,or enzyme cellulase or pectinase. When stressed, however, the cultureproduced up to 3.3 times the normal rate of the ursane-type pentacyclictriterpenes (2% of dry mass) but no increase in the production of indolealkaloids occurred.

[0010] Other experiments with Tabernaemontana divaricata treated withCandida albicans elicitor led to production of a series of pentacyclictriterpenes of the ursane and oleane types, and was accompanied byinhibition of both growth and indole alkaloid accumulation (Van derHeijden et al., (1989) Phytochemistry 28, 2981-1988).

[0011] Also, the tissue culture of Tripterygium wilfordii, normally asource of potent cytotoxic diterpenes, stressed by fungal Botrytiselicitor dramatically enhanced production of oleane-type pentacyclictriterpenes but not the diterpenes, prompting a conclusion that onlytriterpenes are inducible anti-microbial phytoalexins. IKutney, et al.,(1993) Anti-inflammatory oleane triterpenes from Triptervgium wilfordiicell suspension cultures by fungal elicitation. Plant Cell Rep. 12,356-359.

[0012] The conventional treatments for leafy and grassy plants that havebeen attacked by fungi and bacteria are usually exercised after anoutbreak occurs. The affected plants are then treated with one or moreof the commercial synthetic contact antimicrobial sprays at applicationrates that do not pose phytotoxicity concerns. Effective treatment ratesmust be balanced against the risks of harming the treated plant withchemicals that are structurally unrelated to those in the plantphysiology. It would be desirable to have a protective agent that iseffective against microbial pathogens which would also work in a manneranalogous to the plant's natural defense mechanisms to reduce the riskof phytotoxicity. One of the problems associated with treatinghydrophobic leaf surfaces is an effective application of the material.Current spraying techniques result in a portion of the sprayed materialfalling to the ground directly or after being washed off from rainoccurring shortly after application. Either event increases concerns forenvironmental contamination.

[0013] Protective agents that are applied by spraying should remain onthe plant surface for a time sufficient to serve their intendedfunction. Stability against ultraviolet and visible light is, therefore,a concern for foliar treatments. It would be desirable to developfungicides for foliar application that resist degradation by exposure toultraviolet as well as visible light.

[0014] Moreover, invading organisms have been known to evade the effectsof a treatment agent by mutation and propagation of resistant strains.Such developed resistance is economically detrimental because it forcesthe discovery of new treatments. It would be helpful to provide plantanti-infective agents which cannot be evaded by mutating pathogens.

SUMMARY OF THE INVENTION

[0015] It is an object of the invention to provide a composition andprocess for use thereof for protecting plant surfaces against microbialpathogens with ingredients that are compatible with natural plantdefense mechanisms.

[0016] It is another object of the invention to provide a compositionand method of use that poses little risk to the environment, humans, orbeneficial insects.

[0017] It is another object of the invention to provide plantanti-infective agents, which cannot be evaded by developing resistanceby mutating pathogens.

[0018] It is yet another object of the invention to provide acomposition having good resistance to degradation by ultravioletexposure, long storage times, and good inherent sticking ability toplant surfaces.

[0019] In accordance with these and other objects of the invention,which will become apparent from the description herein, a compositionaccording to the invention comprises: a pentacyclic triterpene or amixture of pentacyclic triterpenes, including some plant extracts thatare particularly rich in pentacyclic triterpenes, having as maincomponents compounds described by any of formulas I, II, or III.

[0020] A method according to the invention comprises applying to plantsurfaces an effective amount of a composition comprising a pentacyclictriterpene or a mixture of pentacyclic triterpenes exhibiting any offormulas I, II or III.

[0021] Formulas I, II, and III are:

[0022] wherein:

[0023] R¹=Me, CH₂OH, CH₂OY¹, CH₂O-X-OH, CH₂O-X-OY¹, CH₂O-X-Y²,CH₂O-X-Y³, CH₂NHY, CH₂NY¹ ₂,CH₂Y³, CH₂NH-X-OH, CH₂NH-X-Y², CH₂NH-X-Y³,CH₂NH-X-OY, CH₂O C(O)-OY, CH₂O-X-OY¹, CO₂, COY³, COY², CHO, CH═N(CH₂)_(m)(O(CH₂)_(m))_(n)R⁴, or CH═N(CH₂)_(m)(O(CH₂)_(m))_(n)Y²;

[0024] R², R³═H, OH, OY¹, O-X-OH, O-X-OY¹, O-X-Y², Y³, NHY¹, NY¹ ₂, Y³,NH-X-OH, NH-X-Y², NH-X-Y³, NH-X-OY¹, NY¹-X-OH, NY¹-X-Y², NY¹-X-Y³, orNY¹-X-OY¹;

[0025] protided that one of R² and R³ is H or that R² and R³ togetherdenote carbonyl oxygen;

[0026] R⁴=H, OH, OY, or Y³;

[0027] Y¹=H, alkyl of 1-30 carbon atoms, straight chain or branched,cycloalkyl of 3-30 carbon atoms, alkanyl of 3-30 carbon atoms, oxyalkylof 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms, or phenoxyalkylof 7-30 carbon atoms;

[0028] Y²=NH₂, NHY¹, or NY¹ ₂;

[0029] Y³=—(O(CH₂)_(m))_(n)R⁴ or —(O(CH₂)_(m))_(n)Y², where m=2-4 andn=1-230;

[0030] X=-OC(CH)PCO- where p=1-22.

[0031] The present invention provides a composition and method of usethat are particularly effective in preventing outbreaks of airbornefungal and bacterial diseases on treated plant surfaces. The pentacyclictriterpenes of the composition are applied to form a film over the plantsurface. The film of material prevents the prerequisite attachment ofaerial fungal pathogen and penetration of the plant cuticle. Inaddition, the compounds are not phytotoxic and are compatible withnatural plant defenses

DETAILED DESCRIPTION

[0032] The present invention relates to compositions containing andmethods of using pentacyclic triterpenes having the following formulasI, II, or III:

[0033] wherein:

[0034] R¹=Me, CH₂OH, CH₂OY¹, CH₂O-X-OH, CH₂O-X-OY¹, CH₂O-X-Y²,CH₂O-X-Y³, CH₂NHY¹, CH₂NY¹ ₂,CH₂Y³, CH₂NH-X-OH, CH₂NH-X-Y², CH₂NH-X-Y³,CH₂NH-X-OY¹, CH₂OC(O)—OY¹, CH₂O-X-OY, CO₂Y¹, COY³, COY², CHO,CH═N(CH₂)_(m)(O(CH₂)_(m))_(n)R⁴, or CH═N(CH₂)_(m)(O(CH₂)_(m))_(n)Y²;

[0035] R², R³=H, OH, OY¹, O-X-OH, O-X-OY, O-X-Y², Y³, NHY¹, NY¹ ₂, Y³,NH-X-OH, NH-X-Y², NH-X-Y³, NH-X-OY¹, NY-X-OH, NY-X-Y², NY-X-Y³, orNY-X-OY;

[0036] prouided that one of R² or R³ is H or that R² and R³ togetherdenote carbonyl oxygen;

[0037] R⁴=H, OH, OY¹, or Y³;

[0038] Y¹=H, alkyl of 1-30 carbon atoms, straight chain or branched,cycloalkyl of 3-30 carbon atoms, alkanyl of 3-30 carbon atoms, oxyalkylof 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms, or phenoxyalkylof 7-30 carbon atoms;

[0039] Y²=NH₂, NHY, or NY₂;

[0040] Y³=—(O(CH₂)_(m))_(n)R⁴or —(O(CH₂)_(m))_(n)Y², where m=2-4 andn=1-230;

[0041] X=—OC(CH₂)_(p)CO— where p=1-22.

[0042] The preferred pentacyclic triterpenes include betalin, betuhincacid, ursolic acid, oleanolic acid, betulin mono- and di-succinate orglutarate, as well as polyethylene glycol derivatives of thereof.

[0043] Particularly useful are those pentacyclic triterpenes exhibitingan lC₅₀ value against human leucocyte elastase at concentration lessthan about 15 micromolar (μM), more preferably an IC₅₀ value of lessthan about 10, and most preferably an IC₅₀ value at less than about 8r.nicromolar. The IC₅₀ value represents the concentration of aninhibitor, which can be expressed in micromoles per liter, at whichactivity of an enzyme is reduced by 50%. Thus, lower IC₅₀ values suggesthigher levels of enzyme inhibitory activity.

[0044] The pentacyclic triterpenes of the invention are the same as,derived from, synthesized, or otherwise related to those found naturallyin the outer surfaces of plants: leaves, fruits, bark, and are subjectedto pathogenesis involving enzymatic degradation of cuticle. For thepresent invention, the pentacyclic triterpenes or their derivatives areapplied to the exposed outer plant surfaces in conjunction with asuitable carrier such as water, an aqueous film- forming solution,detergents, emulsion forming additives, suitable polymers to enhancephysical properties of the sprayed layer.

[0045] Pentacyclic triterpenes can be obtained by extracting thepentacyclic triterpene- containing plant tissues with one or moreorganic solvents suitable for the triterpenes. Preferred plant tissuesources for PT include bark from white birch trees, apple peels, and theleaves of plants belonging to Vaccinium and Myristica spp. Usefulsolvents for the extraction include ethyl acetate, acetone, methyl ethylketone, ethanol, propanol, isopropanol, methanol, methylene chloride,chloroform, or their mixtures.

[0046] The pentacyclic triterpenes or their polyethylene glycolderivatives of the invention should be formed in a non-crystalline stateinto a well mixed colloidal suspension for application as a uniformcoating on the treated plant surfaces. The uniform coating helps toensure that plant surfaces are well protected against pathogens with theexception of under surfaces or secluded regions unable to be reached byconventional spraying equipment for liquid formulations.

[0047] The pentacyclic triterpene compounds of the invention are,however, crystalline solids of hydrophobic character. The solids can bedissolved in a number of solvents suitable for agricultural use. Ifdesired, the solutions or colloidal concentrates of pentacyclictriterpenes can be prepared for shipping and storing. This concentratecan then be further diluted for use by a formulator or applicator.

[0048] A preferred solvent for pentacyclic triterpene solids containsabout 1-25 wt % acetone, about 0-10 wt % dimethylsulfoxide (DMSO), 0-35%polyethyleneglycol ester of an aliphatic acid, and about 0-25 wt % of asurfactant such as commercially available detergents like Tween 8™ orPalmolive™dishwashing detergent. Generally, this solvent mixture maycarry a concentration of a PT being 1500-4000%, by weight, of thatneeded for application to the plants.

[0049] The concentrate can be further diluted with 100-4000%, preferably300-1000% by weight of water to make a sprayable composition accordingto the invention. Particularly useful concentrations are within therange from about 7-30 grams per gallon of water. Adequate mixingrequires only low to moderate shearing to ensure adequate mixing of theconcentrate during dilution. For example, metering the concentrate intoa reservoir attached to a venturi nuxer would provide adequate shear tocompletely mix the concentrate with additional water.

[0050] Pentacyclic triterpenes or their polyethylene glycol derivativesaccording to the invention are applied at a rate sufficient to preventpathogenic infections. The inhibitory properties of PT are utilized bythe plants to inactivate the enzymes excreted by the fungal spore inorder to degrade the plant cuticle. Suitable application rates foreffective protection include rates within the range from about 0.1-1000kg/h. Preferably, the application rate is within the range from about0.1-100 kg/h. The specific application rate that is best for aparticular type of plant in a particular region is readily determined bythe application of the ordinary skill in the art. The applicationshould, however, be designed to fall on and cover the exposed leafsurfaces of the plants being treated such as it occurs with conventionalfoliar treatments using conventional foliar spraying equipment.

[0051] If desired, the PT or their polyethylene glycol derivatives maybe applied in conjunction with one or more inert or active ingredients.Exemplary materials include dyes, additives affecting stability of theconcentrate and additives affecting physical properties of the sprayedlayer, foliar fertilizers, fungicides, and insecticides.

[0052] Virtually any plant that may get infected through the waxycuticle layer can be beneficially treated with compositions according tothe present invention. Commercial plants that would benefit includegrain grasses (e.g., rye, wheat, and barley), tomato, bean, pepper,wheat, and peanut plants.

[0053] Grain grasses may benefit in particular from the presentinvention. These plants do not generally produce sufficient levels ofpentacyclic triterpenes, if at all, to inhibit enzymatic attacks byinvading microorganisms. Natural pathogens of these plants are generallynot adapted to produce sufficient amounts of enzymes to overcome theinhibiting effects of the externally applied pentacyclic triterpenecompounds.

[0054] Plants susceptible to small insect (e.g., aphids) infestationalso benefit from treatment according to the invention. Ursolic acidshowed toxicity and feeding deterrent effects towards the mites andtheir survival. The mites' reproductive indexes decreased in directproportion to ursolic acid content in the diet, and in addition,ingestion time on diet containing ursolic acid was reduced about 30%(Varanda et al., 1992). These observations imply a possibility that theinsects digestive enzymes were compromised by ursolic acid.

[0055] The mode of inhibition of enzymes by PT is non-specific and isbased primarily on hydrophobic interaction with an enzymes hydrophobicdomain. This property suggests that the likelihood for developingmicrobial pathogen resistance through mutation is remote. Pathogens hadto deal with the presence of PT in the plant cuticle for as long as theplants existed and it could be assumed that microbial resistance to thePT has been already optimized. The PT based formulations for plantprotection should be advantageous against the pathogenic microorganismsthat use enzymes to break through the cuticle, such as fungi, bacteria,nematodes and viruses. These formulations should be also advantageousagainst small insects, acting inhibitory on their digestive enzymes. Bycovering the leafy surface with a film containing pentacyclictriterpenes, the passive defensive properties of the cuticle areenhanced, which decreases or entirely prevents successful pathogenicpenetration.

[0056] The following examples are included to assist in an understandingof the invention and are not intended to limit the scope of the attachedclaims.

EXAMPLES

[0057] Commercially available compounds were acquired either fromExtrasyntheses (France), Aldrich, Sigma or ICN Pharmaceuticals. Meltingpoints were obtained using a Reichert Thermovar melting point apparatusand are corrected. Hnmr spectra were acquired in C₅D₅N with TMS as aninternal standard (δ=0.0 ppm) using a Bruker WM-250 or 270 NMRspectrometer. Mass spectra were acquired using a Finnigan 4023 El/ClGCMS mass spectrometer with Super-INCOS software and direct insertionprobe.

Examples 1a and 1b Synthesis of 28-hemisuccinylbetulin and3,28-Di-hemisuccinylbetulin Example 1a

[0058] A reaction mixture is prepared by dissolving imidazole (4 eq) andsuccinic anhydride (1.08 eq) in N-methylpyrrolidone. The volume of thesolvent is about 4.5 times the weight of betulin. When the two reagentsare dissolved, betulin (1 eq) is added. The reaction mixture is left atroom temperature with occasional shaking. After approximately 48 hrs,about 92-95 % of the betulin has been converted into28-hernisuccinilbetulin, with 2-4 % as unconverted betulin anddiacvlbetulin.

[0059] The 28-hetmisuccinilbetulin is purified by pouring the reactionmixture into a 20X volume of water with slow stirring. The water isdecanted from the solid residue and the washing is repeated with severalnew portions of water. The solid is separated, dried and dissolved in amixture of chloroform and isopropanol. The chloroform is removed byevaporation enabling 28-hemisuccinylbetulin to form and crystallize. The28-hemisuccinylbetulin crystals are white with a melting point of242-244° C.

[0060] Calculations for C₃₄H₅₄O₅, C, 75.22%, H, 10.03; found C, 75.0, H,9.91. The ¹Hnmr (DMSO-d₆) gave peaks at: 4.86 and 4.72 (1H, s, each,CH₂═C), 4.57 and 4.14 (AB pattern, J=12 Hz, OCH₂O), 3.44 (1H, t,J=8.6Hz), 2.95 (4H, s, CHOCH₂CH₂CO), 1.71, 1.22, 1.02, 25 0.98, 0.98, 0.84(s, each, Me); eits (m/z, rel. int.): 524 (20, [M-H₂O]+), 424 (15), 411(27), 207 (38), 203 (41), 189 (100), 135 (70), 119 (61), 107 (62), 95(81), 81 (79), 55(90%); cims: 543 (11, M+), 525(64, [MH-H₂O]+), 425(80),407 (100), 217 (91), 203 (90), 191 (48), and 189 (52%).

Example 1b

[0061] Betulin, 443 mg (1 mmole), imidazole, 227 mg (4 mmoles), succinylanhydride, 400 30 mg (4 rmmoles) and 0.6 ml of methylpyrrolidone werecombined in a vial. The reaction mixture was maintained at 70° C. for 20hts. The reaction mixture was then added dropwise to 100 ml of water andstirred for half an hour. The resulting precipitate was filtered off,re-suspended and agitated in another portion of water in order to removethe remaining reactants. This procedure was repeated several times untilno more of the reactants were washed out. The solid was collected, driedand dissolved in acetone. The acetone solution was filtered andevaporated to give 470 mg of 3,28-dihemisuccinylbetulin.

[0062] Calculated for: C₃₈H₅₈O₈·¼ H₂O, MW 642.84; C, 70.5%, H, 9.1%;found C 70.43%, H 9.15%. 1H NMR: 4.70 and 4.61 (1H, s, each, =CH₂), 4.52(1H, dd,J₁=5.4 Hz,J₂=10.0 Hz), 4.33 and 3.90 (1H, d, each, AB pattern,J=11.1 Hz, OCH₂), 2.63-2.71 (8H, m, succinyl chain), 1.70, 1.05, 0.99,0.87, 0.86, and 0.85 (Me, s, each). MALDI-TOF-MS gave m/z 665 (M+Na⁺)and also m/z 542 and 525.

Example 2 Synthesis of Acetylbetulinic Acid

[0063] Betulinic acid, 100 mg (0.22 nmioles) was added to a solutioncontaining 4 mg of 4-dimethylaminopyridine (DMAP) and 0.5 ml of(CH₃CO)₂O in 5 ml of CH₂Cl₂. After 2 hrs CH₂Cl₂ was removed in iacuo,and the remaining residue stirred with 25 ml of water. During thestirring enough of K₂CO₃ was added to decompose an excess of aceticanhydride, after which the reaction mixture was extracted with 25 ml ofCH₂Cl₂. The extract was evaporated to dryness and the resulting solidcrystallized from MeOH to give 104 mg of acetylbetulinc acid as whitecrystals, m.p. 285° C.

[0064] Calculations for C₃₂H₅₀O₄, C, 77.06, H, 10.10; found C, 77.02, H,10.08%. The ¹Hnmr (DMSO-d6) gave peaks at: 4.93 and 4.76 (1H, s, each,CH₂=C), 4.67 (1H, dd, C3-H, J=5.0, 10.0 Hz), 2.05 (s, CH₃CO), 1.78,1.06, 1.00, 0.87, 0.84 and 0.73 (each, s, CH₃); eims (m/z, relativeintensity): 499 (1, MH+), 452 (2), 438 (38), 395 (26), 189 (40), 43(100%); cims (m/z, rel. intensity): 499 (37, MH+), 440 (27), 439 (100),437 (18), 393 (20), 203 (15), 191 (24%).

Example 3 Synthesis of 3-Methanesulfonylbetulinc Acid

[0065] Betulinic acid, 100 mg (0.22 mM) was dissolved in 5 ml ofpyridine and treated with 100 mg (0.88 mmoles) of CH₃SO₂Cl. After 16hrs, the pyridine was removed in vacuo and the resulting residuesuspended in 10 ml of water. Excess of MeSO₂Cl was decomposed with anaqueous NaHCO₃ solution, and the reaction mixture extracted with 10 rmlof CH₂C1₂. Evaporation of the solvent gave a crude product containing3-methanesulfonylbetulinic acid, which was purified by columnchromatography over 3 g of silica gel using CH₂CI₂ with 0 to 5% gradientof MeOH. The chromatography afforded 3-methanesulfonylbettnic acid,which on crystallization from MeOH gave 62 mg of white crystals, m.p.210-212° C.

[0066] Calculations for C₃₁H₅₀O₅S: C, 69.62, H, 9.42, S, 6.00; found C,69.78, H, 9.46, S, 5.90%. ¹Hnmr (DMSO-d₆): 4.95 and 4.78 (1H, each, s,CH₂=C), 4.50 (1H, dd,J 4.6, 11.8 Hz), 3.30 (3H, s, CH₃SO), 1.79, 1.07,1.06, 1.00, 0.82, 0.72 (each, s, CH₃); eims (m/z, relative intensity):438 (9), 423 (11), 395 (72), 259 (12), 161 (26), 135 (62), 121 (100),107 (60), 93 (61), 79 (540/%); cims (m/z, rel. intensity): 535 (11,MH+), 439 (91), 423 (25), 395 (52), 249 (12), 203 (28), 191 (34), 97(100%).

Example 4 Synthesis of the Methyl Ester of 3-MethanesulfonylbetulinicAcid

[0067] Betulinic acid methyl ester, 500 mg (1.06 mmoles), and 320 mg(3.3 mmoles) of Me₃N were dissolved in 10 ml of CH₂Cl₂. The solution wascooled in an ice-bath and treated with 190 rg (1.7 mrtnoles) of MeSO₂Cl.The reaction mixture was allowed then to warm up to room temperature andleft overnight. Evaporation of the solvent in racuo left a residue,which was dissolved in 10 ml of Et₂O and washed 3 times with 5 ml H₂0.The etheral layer was dried over MgSO₄, filtered, and evaporated to give582 mg of the methyl ester of 3- methanesulfonylbetulnic acid.Recrystallization from EtOH gave crystals with a melting point of 190°C.

[0068] Calculation for C₃₂H₅₂O₅S: C, 70.03, H, 9.55, S, 5.84; found C,69.85, H, 9.50, S, 5.65%. Hnmnr DMSO-d₆): 4.89 and 4.73 (1H, each,CH₂=C), 4.47 (1H, dd, C3-H,J=2.5, 12.5 Hz), 3.70 (s, OCH₃), 3.29 (s,CH₃SO₂), 1.72, 1.04, 0.99, 0.92, 0.81, 0.72 (s, each, CH₃); eims (m/z,relative intensity): 452 (9), 409 (14), 341 (8), 273 (15), 255 (12), 189(100), 175 (47), 121 (72), 107 (72), 93 (76), 79 (850/); cims (m/z, rel.intensity): 549 (16%, MH+), 453 (100), 393 (19), 203 (13), 189 (12), 97(80%).

Examples 5-24 Elastase Inhibition

[0069] Studies, performed with human leucocyte elastase (HLE) indicatedthat many common naturally occurring PTs with lupane, oleane, and ursaneskeletons inhibit HLE at low micromolar concentrations. Severalderivatives of these PTs were prepared using synthetic methodology toexplore structure-activity relationship and on testing they alsoinhibited HLE. The values of inhibitory constants, IC₅₀'s obtained fromHLE inhibition, for the tested compounds, are given in Table 1.

[0070] Stock solutions were mixed to yield a reaction mixture consistingof 0.5 mM MeO-Suc-Ala-Ala-Pro-Val-pNA (Sigma) and 0.7 μmg/ml humanleucocyte elastase (Elastin Products) in 0.1 M Tris, 0.5 M NaCl and 3%DMSO (v/v) at a pH of 7.5. Test compounds were solubillzed in DMSO.Equal volumes of enzyme and inhibitor were mixed and allowed to incubatefor 15 minutes at room temperature. The reaction was started by theaddition of a third equal volume containing substrate. Reactions werecarried out in microliter plates. Appearance of p-nitroaniline wasmonitored at 405 nm in a Dynatech mnicroelisa Reader M600. Initial ratesin the presence and absence of test compound were compared to determineIC₅₀ values which are reported in Table 1. TABLE 1 Ex. Compound NameIC₅₀ (μM) 5 Lupeol 8.4 6 Lupeol acetate 14.9 7 Betulin 5.0 828-Succinylbetulin 6.3 9 Betulinic acid 3.3 10 Methyl ester of betulinicacid 5.7 11 Acetylbetulinic acid 7.0 12 3-Ketobetulinic acid 11.0 13Methanesulfonylbetulinic acid 11.2 14 Methyl ester of3-methanesulfonylbetulinic acid 7.3 15 β-Amyrin 5.4 16 Uvaol 4.5 17Ursolic acid 7.7 18 Ursolic acid, Me ester 4.9 19 α-Amyrin 21.1 20Oleanolic acid 18.7 21 Oleanolic acid, Me ester 8.5 22 Echinocystic acid21.1 23 Hederagenin 16.8 24 Caulophyllogenin 0% @ 21°

[0071] Further experiments, carried out with ursolic acid and oleanolicacid, indicated that these compounds can inhibit also other enzymes,such as plasmin and urokinase. The values of inhibition, measured at 22μM in the plasmin assay were 43% for ursolic acid and 25% for oleanolicacid, while in the urokinase assay were 83% for ursolic acid and 40% foroleanolic acid. For betulinic acid, in the following assays, lC₅₀ valueswere determined to be: thrombin 2.3, trypsin 8.5, plasmin 6.2, andurokinase 5.0 μM. The apparent lack of specificity within the testedenzymes and an observation that the inhibition ceases in the presence of0.1% of albumin suggested that the inhibition results from anon-specific binding to proteins. Thus, PTs appear to bindnon-covalently to hydrophobic domains of enzymes and either block anaccess to the enzyme active site or cause conformational change, bothresulting in the enzymes inability to perform its function.

[0072] The most potent inhibitors of the investigated group of PTscontain one to three oxygenated substituents, such as hydroxy or carboxygroups. These moieties contribute to the overall inhibitory effect ofthe compounds.

Examples 25 Fungicidal Activity

[0073] Tomato, bean, pepper, wheat, and peanut plants are grown for oneto three weeks (depending upon species) in the greenhouse. Two pots,which represent two replicates, of each plant species are placed into aflat such that each flat contains all the plants to be sprayed by onecompound. The plants in each flat are sprayed to runoff with the desiredspraying solution or with fungicide standard. As a control, check plantsare sprayed with water. The plants are allowed to air-dry two to threehours. After the plants are dry, they are sorted and grouped by plantspecies.

[0074] Table 2 identifies the compositions of examples 25-28 that weretested for their ability to protect hydrophobic plant surfaces: TABLE 2Example Material 25 Control vehicle containing acetone 18 ml,dimethylsulfoxide 4 ml, and Palmolive ™ detergent 200 mg. Tested as 3%aqueous concentration. 26 40 mg betulin dissolved in 1.5 ml of vehicleand added to 98.5 ml of water. 27 80 mg betulin, 3.0 ml of vehicle, and97 ml of water. 28 40 mg of ursolic acid, 2.0 ml of vehicle, and 98 mlof water.

[0075] The plant pathogenic fungi Phytophthora infestans (Pi),Alternaria solani (As), Botryds cinerea (Bc), and Cercosporaarachidicola (Ca) were grown in the laboratory on appropriate media.Inoculum from each fungus was harvested and concentrations adjusted topredetermined levels. The obligate plant pathogenic fungi (Ervsiphegraminis sp. tritici, Puccinia recondita f sp. tritici) are harvestedfrom their hosts in the greenhouse and concentration are adjusted topredetermined levels.

[0076] The plants previously treated with test compounds are sprayedwith fungal inoculum then placed in humidity chambers for a period oftime which is found to be optimum for development of each disease. Afterincubation, the plants are moved to the greenhouse, symptoms allowed todevelop, and the plants evaluated for disease intensity. Table 3 reportsthe percent disease control as the average of two replicates. TABLE 3Disease (% control) Example Pi Pv As Bc Sn Pr Eg 25  8 phytotoxic  5 120 0 0 26  0 0 17  3 0 0 0 27 40 0  3  7 0 0 0 28 65 phytotoxic  8 33 0 00 29 33 0 12 12 0 0 0

[0077] Following review of the control achieved by examples 25-29, theconcentration of pentacycic triterpene and carrier vehicle were changed.Table 4 identifies the compositions of examples 30-35, and Table 5 showsthe percent control of plant diseases following a protective applicationof materials in examples 30-35. The results in table 5 are the mean ofthree replicates. TABLE 4 Example Composition 30 Carrier vehiclecontaining 0.35% solution of Tween 80 ™ in water. 31 200 mg betulin in100 ml of carrier. 32 400 mg betulin in 100 ml of carrier. 33 800 mgbetulin in 100 ml of carrier. 34 400 mg ursolic acid in 100 ml ofcarrier.

[0078] TABLE 5 Disease (% control) Ex. Pi Pv As Bc Sn Pr Eg Sm-p Sm-t 307 0 43 94 0 0 0 50 30 31 10  0 47 100  0 0 0 60 20 32 7 37  53 82 0 0 065 30 33 60  0 63 97 0 0 0 50 30 34 0 0  0 60 0 0 0 40 60

[0079] As seen from Table 5, an increase in the amount of appliedbetulin generally increased control in susceptible fingus.

1. A composition useful for inhibiting enzyme-based attacks on plantsurfaces comprising: a carner selected from the group consisting ofaqueous fiin-forming solutions, dimethylsulfoxide, ethyl acetate,acetone, methyl ethyl ketone, methylene chloride, chloroform, andmixtures thereof, and a pentacyclic triterpene compound exhibiting astructure of formula II:

wherein: R¹=Me, CH₂OH, CH₂OY, CH₂O-X-OH, CH₂O-X-OY¹, CH₂O-X-Y²,CH₂O-X-Y³, CH₂NHY, CH₂NY¹ ₂,CH₂Y³, CH₂NH-X-OH, CH₂NH-X-Y², CH₂NH-X-Y³,CH₂NH-X-OY¹, CH₂OC(O)-OY¹, CH₂O-X-OY¹, CO₂Y¹, COY³, COY², CHO,CH═N(CH₂)m(O(CH2,),R⁴, or CH═N(CH₂)_(m)(O(CH₂)_(m) )_(n))Y²; R², R³=H,OH, OY¹, O-X-OH, O-X-OY¹, O-X-Y², Y3, NHY¹, NY₂, Y3, NH-X-OH, NH-X-Y²,NH-X-Y³, NH-X-OY¹, NY-X-OH, NY¹-X-Y², NY¹-X-Y³, or NY-X-OY¹; providedthat one of R² or R³ is H or that R² and R³ together denote carbonyloxygen; R⁴=H, OH, OY, or Y³; Y¹=H, alkyl of 1-30 carbon atoms, straightchain or branched, cycloalkyl of 3-30 carbon atoms, alkanyl of 3-30carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbonatoms, or phenoxyalkyl of 7-30 carbon atoms; Y²=NH₂, NHY¹, or NY¹ ₂;Y³=—(O(CH₂)_(m))_(n)R⁴ or —(O(CH₂)_(m))_(n)Y², where m=2-4 and n=1-230;X=−OC(CH₂)_(p)O where p=1-22.
 2. A composition according to claim 1further comprising a dye, foliar fertilizer, fungicide, or insecticide.3. A composition according to claim 2 further comprising a foliarfertilizer, fungicide, or insecticide.
 4. A grain grass having externalsurfaces covered by a composition according to claim 1 .
 5. A graingrass according to claim 4 selected from the group consisting of rye,wheat, or barley.
 6. A method for protecting plants against fungusattack by a process comprising: applying to exposed plant surfaces acarrier and an effective amount of a composition containing apentacyclic triterpene compound exhibiting a structure according toformula II:

wherein: R¹=Me, CH₂OH, CH₂OY¹, CH₂O-X-OH, CH₂O-X-OY¹, CH₂O-X-Y²,CH₂O-X-Y³, CH₂NHY¹, CH₂NY¹ ₂,CH₂Y³, CH₂NH-X-OH, CH₂NH-X-Y², CH₂NH-X-Y³,CH₂NH-X-OY, CH20C(O)-OY¹, CH₂O-X-OY¹, CO₂Y¹, COY³, COY², CHO,CH═N(CH₂)_(m)(O(CH₂)_(m)R⁴,or CH═N(CH₂)_(m)(O(CH₂)₂Y²; R², R³=H, OH,OY¹, O-X-OH, O-X-OY¹, O-X-Y², Y³, NHY¹, NY², Y³, NH-X-OH, NH-X-Y2,NH-X-Y³, NH-X-OY¹, NY-X-OH, NY¹-X-Y², NY¹-X-Y³, or NY¹-X-OY;promded thatone of R² and R³ is H or that R² and R³ together denote carbonyl oxygen;R⁴=H, OH, OY¹, or Y³; Y¹=H, alkyl of 1-30 carbon atoms, straight chainor branched, cycloalkyl of 3-30 carbon atoms, alkanyl of 3-30 carbonatoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms,or phenoxyalkyl of 7-30 carbon atoms; Y²=NH₂, NHY¹, or NY¹ ²;Y³=—(O(CH₂)_(m))_(n)R⁴ or —(O(CH₂)_(m))_(n)Y², where m=2-4 and n=1-230;X=—OC(CH₂)_(p)CO— where p 1-22.
 7. A method as in claim 6 wherein theapplying step comprises applying said composition to surfaces of graingrasses.
 8. A method as in claim 6 wherein said carrier is selected fromthe group consisting of an aqueous film-formig solution, a surfactant,an emulsion formig additive, and a polymer.
 9. A method as in claim 6wherein said carrier is selected from the group consisting of aqueousfilm-forming solutions, dimethylsulfoxide, ethyl acetate, acetone,methyl ethyl ketone, methylene chloride, chloroform, and mixturesthereof.
 10. A method as in claim 6 wherein said composition contains asolvent for said pentacyclic triterpene compound.
 11. A method as inclaim 10 wherein said solvent is selected from ethyl acetate, acetone,methyl ethyl ketone, ethanol, propanol, isopropanol, methanol, methylenechloride, chloroform, or their mixtures.
 12. A method according to claim10 wherein said solvent contains 1-25 wt % acetone, about 0-10 wt %dimethylsulfoxide, 0-35% polyethyleneglycol ester of an aliphatic acid,and about 0-25 wt % of a surfactant.
 13. A method as in claim 6 whereinsaid pentacyclic triterpene compound is applied to said plant surfacesat a rate sufficient to prevent pathogenic infections.
 14. A method asin claim 13 wherein said pentacyclic triterpene compound is applied tosaid plant surfaces at a rate within the range of 0.1-1000 kg/h.
 15. Amethod as in claim 14 wherein said pentacyclic triterpene compound isapplied to said plant surfaces at a rate within the range of 0.1-100kg/h.